Application of virus detection system in preparation of children respiratory pathogen detection kit and kit thereof

Abstract

The invention provides application of a virus detection system in preparation of kits for detecting respiratory pathogens of children, the system comprises a kit for simultaneously detecting influenza A virus, influenza B virus, new coronavirus and bocavirus, and the system comprises a kit for simultaneously detecting influenza A virus, influenza B virus, new coronavirus and enterovirus; the system comprises a kit for simultaneously detecting mycoplasma pneumoniae, pertussis, metapneumovirus and moraxella catarrhalis, the system comprises a kit for simultaneously detecting the rhinovirus, the respiratory syncytial virus, the adenovirus and the mycoplasma pneumoniae; according to the big data analysis result, the typical symptoms of the respiratory tract infection of the children are divided into four types, namely pure continuous hyperpyrexia, pure severe cough, severe cough and hyperpyrexia and pure wheeze, and a plurality of pathogens which are preferentially detected are set for each type of symptoms, so that the detection efficiency can be improved, and the waste of medical resources is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to use of a virus detection system in preparing a children's respiratory pathogen detection kit and a kit thereof. Background Art

[0002] Respiratory tract infection is one of the common diseases in children. Children have poorer immunity than adults, so respiratory tract infections in children usually have higher morbidity and mortality, and often require hospitalization. Respiratory tract infections in infants and young children are prone to go down, causing complications and involving adjacent organs and tissues. Acute lower respiratory tract infections may cause greater harm to children. Worldwide, acute lower respiratory tract infection is the leading cause of death in children under 5 years old, far higher than other causes of death such as diarrhea and accidental suffocation. Therefore, in the early stages of respiratory tract infection, accurate diagnosis of pathogens and early intervention are of great significance. Children's upper respiratory tract infections usually show clinical symptoms such as fever, runny nose, cough, and difficulty swallowing. Depending on the location of the occurrence, they are clinically diagnosed as acute pharyngitis, acute tonsillitis, acute nasopharyngitis (cold), acute pharyngococcygitis, etc. Studies have observed that children of different ages with different respiratory tract infection symptoms are infected with different pathogens.

[0003] Common detection methods for respiratory pathogens include isolation and culture, antigen detection, antibody detection, and nucleic acid detection. Isolation and culture are time-consuming and laborious, and the positive rate is not high (due to the difficulty of virus culture and the inability to culture some pathogens); antigen detection has low sensitivity and is prone to missed detection; antibody detection has a long window period and has relatively little value for early diagnosis of most respiratory pathogen infections. Nucleic acid detection has the advantages of high sensitivity and strong specificity, and has great value for early infection diagnosis, which is conducive to early detection, early isolation, early reporting, and early treatment of respiratory infections. Therefore, nucleic acid detection is becoming more and more widely used in clinical practice. The purpose of the present invention is to develop a set of multiplex qPCR kits for detecting common respiratory pathogens in children, covering 15 common pathogens of children's respiratory infections, including respiratory syncytial virus RSV, influenza A virus INF-A, parainfluenza virus PIV, new coronavirus nCOV, influenza B virus INF-B, rhinovirus HRV, Mycoplasma pneumoniae MP, adenovirus ADV, human bocavirus hBoV, human enterovirus EnV, human metapneumovirus MPV, Streptococcus pneumoniae SP, Moraxella catarrhalis MC, Bordetella pertussis BP, and Haemophilus influenzae Hi. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a use of a virus detection system in preparing a children's respiratory pathogen detection kit, the system comprising:

[0005] When the clinical symptom of the child is simple persistent high fever and the child is not less than 2 years old, the system includes a test kit for simultaneously detecting influenza A virus, influenza B virus, new coronavirus, and boca virus;

[0006] When the child's clinical symptoms are simple persistent high fever and the child is less than 2 years old, the system includes a test kit for simultaneously detecting influenza A virus, influenza B virus, new coronavirus, and enterovirus;

[0007] When the clinical symptom of the child is a simple severe cough and the child is not less than 5 years old, the system includes a test kit for simultaneously detecting Mycoplasma pneumoniae, pertussis, metapneumovirus, and Moraxella catarrhalis;

[0008] When the child's clinical symptom is a simple severe cough and the child is less than 5 years old, the system includes a test kit for simultaneously detecting rhinovirus, respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae;

[0009] When the clinical symptoms of the child are high fever with severe cough, and the child is not less than 3 years old, the system includes a test kit for simultaneously detecting Mycoplasma pneumoniae, Streptococcus pneumoniae, parainfluenza virus, and Haemophilus influenzae;

[0010] When the clinical symptoms of the child are high fever with severe cough, and the child is less than 3 years old, the system includes a test kit for simultaneously detecting respiratory syncytial virus, adenovirus, Streptococcus pneumoniae, and metapneumovirus;

[0011] When the child's clinical symptom is wheezing and the child is not less than 5 years old, the system includes a test kit for simultaneously detecting Mycoplasma pneumoniae, adenovirus, and Moraxella catarrhalis;

[0012] When the child's clinical symptom is wheezing and the child is less than 5 years old, the system includes a test kit for simultaneously detecting respiratory syncytial virus, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae.

[0013] In one embodiment, the children's respiratory pathogen detection kit is a fluorescent quantitative PCR detection kit.

[0014] In one embodiment, the primers and probes for each pathogen in the detection system are as follows:

[0015]

[0016]

[0017] In one embodiment, the kit is used when the clinical symptom of the child is simple persistent high fever and the age is not less than 2 years old. The kit is a kit for detecting influenza A virus, influenza B virus, new coronavirus, and boca virus at the same time, and the kit includes the following primers and probes:

[0018] ,

[0019] When the child's clinical symptoms are simple persistent high fever and the child is less than 2 years old, the system is a kit for simultaneously detecting influenza A virus, influenza B virus, new coronavirus, and enterovirus, and the kit includes the following primers and probes:

[0020]

[0022] In one embodiment, a kit for use in the above-mentioned purpose is provided, wherein the kit is used when the clinical symptom of the child is simple severe cough and the age is not less than 5 years old, and the system is a kit for simultaneously detecting Mycoplasma pneumoniae, pertussis, metapneumovirus, and Moraxella catarrhalis; the kit comprises the following primers and probes:

[0023] ,

[0024] When the clinical symptom of the child is simple severe cough and the child is less than 5 years old, the system is a kit for simultaneously detecting rhinovirus, respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae; the kit includes the following primers and probes:

[0025]

[0026] In one embodiment, the kit is used when the clinical symptoms of a child are high fever with severe cough and the child is not less than 3 years old. The kit is a kit for simultaneously detecting Mycoplasma pneumoniae, Streptococcus pneumoniae, parainfluenza virus, and Haemophilus influenzae. The kit includes the following primers and probes:

[0027] ,

[0028] When the clinical symptoms of children are high fever with severe cough, and the age is less than 3 years old, the system is a kit for simultaneously detecting respiratory syncytial virus, adenovirus, Streptococcus pneumoniae, and metapneumovirus, and the kit includes the following primers and probes:

[0029]

[0030] In one embodiment, the kit is used when the child's clinical symptom is wheezing and the child is not less than 5 years old. The kit is a kit for simultaneously detecting Mycoplasma pneumoniae, adenovirus, and Moraxella catarrhalis, and the kit includes the following primers and probes:

[0031]

[0032] The kit is used when the clinical symptom of the child is wheezing and the child is less than 5 years old. The kit is a kit for simultaneously detecting respiratory syncytial virus, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae. The kit includes the following primers and probes:

[0033]

[0034] Based on the results of big data analysis, the present invention divides the typical symptoms of children's respiratory tract infections into four categories: simple persistent high fever, simple severe cough, severe cough and high fever, and simple wheezing, and sets several pathogens for priority detection for each type of symptom, which can improve detection efficiency and avoid waste of medical resources.

[0035] At the same time, the present invention has developed a primer and probe combination for detection using the qPCR method for 15 common respiratory pathogens in children. The present invention designs 15 sets of primer probes. The primer probes in each tube of the 15 pathogens in the 3-4-plex PCR reaction according to the pathogen combination can achieve the most stable dimer entropy value dG exceeding -7kcal / mol, which can avoid cross-reactions between primers and probes in the same reaction tube. DETAILED DESCRIPTION

[0036] In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0037] Example 1 Analysis of respiratory tract infection viruses in children

[0038] The present invention includes 53,347 outpatients and inpatients who were tested for respiratory pathogens in Guangdong Provincial Women's and Children's Health Care Hospital for a total of five years from 2017 to 2019 and 2023 to 2024. A total of 15 major respiratory pathogens were detected. The detection methods included antibody detection, antigen detection, qPCR detection, liquid chip detection, tNGS targeted sequencing, etc. All patients were divided into four major symptoms according to the description of the medical records (. The results showed that the typical symptoms of respiratory tract infection in children were divided into four categories: simple persistent high fever, simple severe cough, severe cough and high fever, and simple wheezing. According to clinical observations and test results, the detection rates of highly prevalent pathogens corresponding to children of different ages and symptoms were also different, see Table 1 and Table 2.

[0039] Table 1 Respiratory pathogens detected in children in Guangdong Provincial Women and Children’s Health Care Hospital from 2017 to 2019 and from 2023 to 2024

[0040]

[0041]

[0042] The 15 common pathogens of children's respiratory tract involved in the present invention include: respiratory syncytial virus RSV, influenza A virus INF-A, parainfluenza virus PIV, new coronavirus nCOV (data from 2023-2024), influenza B virus INF-B, rhinovirus HRV, Mycoplasma pneumoniae MP, adenovirus ADV, human bocavirus hBoV, human enterovirus EnV, human metapneumovirus MPV, Streptococcus pneumoniae SP, Moraxella catarrhalis MC, Bordetella pertussis BP, Haemophilus influenzae Hi. The cases include cases where the children are co-infected with several pathogens.

[0043] Based on the above five years of children's respiratory pathogen detection data, the corresponding high-incidence pathogens of respiratory tract infections in children of different ages and with different symptoms were obtained, so as to achieve targeted detection. See Table 2 for details.

[0044] Table 2 Common pathogens for respiratory infections in children of different ages and symptoms

[0045]

[0046] Example 25 Design of primers and probes for pathogens

[0047] The present invention also developed a primer and probe combination for the above 15 common respiratory pathogens in children using the qPCR method for detection. The present invention uses the Taqman probe method, which is currently the most stable in clinical operation and interpretation, for multiplex PCR detection, and is suitable for various types of fluorescent quantitative PCR instruments currently most commonly used in clinical practice. At present, mainstream fluorescence quantitative PCR instruments can achieve 4 fluorescent labels in a single tube. Therefore, in order to achieve the detection of 15 pathogens, the present invention designs primers and probes with the goal of being able to perform 4-plex qPCR reactions simultaneously. The primer and probe design follows the following principles: the primers and probes of each pathogen are retrieved by NCBI and specifically target the target pathogens; the primers and probes in the 3-4-plex PCR reactions of each tube of the 15 pathogens according to the above pathogen combination have been repeatedly calculated by the MFEprimer v4.0 online tool (https: / / m4.igenetech.com / dimer / ) (reference Wang, K. et al., MFEprimer-3.0: quality control for PCRprimers. Nucleic acids research 47, W610–W613 (2019).), and finally the entropy value dG of the most stable dimer is more than -7 kcal / mol, which can avoid cross-reaction of primers and probes in the same reaction tube. The primer-probe combination we finally used is shown in Table 3. The dimer situation of the primer-probe combination for pathogens with high detection rates is shown in Table 4.

[0048] Table 3 Primer probes designed for 15 pathogens

[0049]

[0050]

[0051] Table 4 Dimerization of primer-probe combinations for pathogens with high detection rates

[0052]

[0053]

[0054] The primer probes of the above 15 pathogens can be combined into a tube for detection in up to 4 combinations, and the probes are labeled with FAM, VIC, ROX, and CY5 respectively; if each tube detects only <= 3 pathogens, a human internal reference GAPDH gene can also be added to the tube. The reaction system uses the qRT-PCR premix reaction system produced by Novozymes, and the specific reaction system for each tube is:

[0055]

[0056] Add water to 20 μl.

[0057] The reaction procedure is as follows:

[0058]

[0059] Fluorescence was collected at 60°C. When the Ct value of the target pathogen was <38, the pathogen was considered positive.

[0060] Example 3 Application of the virus detection system of the present invention

[0061] The 4-tube 4-plex PCR designed for the above 15 fragments was used for 570 children who came to the hospital for respiratory tract infection. They were grouped according to their clinical manifestations at the time of the visit and tested for respiratory pathogens. When the Ct of each pathogen was less than 38, the fragment was judged to be pathogen positive. The results of the high-incidence pathogens detected by different ages and symptoms are shown in the following table.

[0062] Table 5. Detection rate of pathogens of simple persistent high fever

[0063]

[0064]

[0065] From the above results, it can be seen that in children with respiratory tract infection with simple persistent high fever as the main symptom, the pathogens detected using the multiplex qPCR detection kit of the present invention are consistent with the highly prevalent pathogens marked in the present invention.

[0066] Table 6. Detection rate of pathogens in simple severe cough

[0067]

[0068] From the above results, it can be seen that in children with respiratory tract infection with simple severe cough as the main symptom, the pathogens detected using the multiplex qPCR detection kit of the present invention are consistent with the highly prevalent pathogens marked in the present invention.

[0069] Table 7. Detection rate of pathogens in patients with high fever and severe cough

[0070]

[0071] From the above results, it can be seen that in children with respiratory tract infection with high fever and severe cough as the main symptoms, the pathogens detected using the multiplex qPCR detection kit of the present invention are consistent with the highly prevalent pathogens marked in the present invention.

[0072] Table 8. Detection rate of pathogens in simple wheezing

[0073]

[0074] From the above results, it can be seen that in children with respiratory tract infection with simple wheezing as the main symptom, the pathogens detected using the multiplex qPCR detection kit of the present invention are consistent with the highly prevalent pathogens marked in the present invention.

[0075] Based on the above results, the present invention provides corresponding combinations of highly prevalent pathogens according to the different ages and symptoms of patients, and develops convenient and effective 15 pathogen qPCR primer probe combinations and detection kits for these highly prevalent pathogen combinations.

[0076] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0077] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. Use of a virus detection system in the preparation of a children's respiratory pathogen detection kit, characterized in that: The system comprises: When the clinical symptom of the child is simple persistent high fever and the child is not less than 2 years old, the system includes a test kit for simultaneously detecting influenza A virus, influenza B virus, new coronavirus, and boca virus; When the child's clinical symptoms are simple persistent high fever and the child is less than 2 years old, the system includes a test kit for simultaneously detecting influenza A virus, influenza B virus, new coronavirus, and enterovirus; When the clinical symptom of the child is a simple severe cough and the child is not less than 5 years old, the system includes a test kit for simultaneously detecting Mycoplasma pneumoniae, pertussis, metapneumovirus, and Moraxella catarrhalis; When the child's clinical symptom is a simple severe cough and the child is less than 5 years old, the system includes a test kit for simultaneously detecting rhinovirus, respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae; When the clinical symptoms of the child are high fever with severe cough, and the child is not less than 3 years old, the system includes a test kit for simultaneously detecting Mycoplasma pneumoniae, Streptococcus pneumoniae, parainfluenza virus, and Haemophilus influenzae; When the child's clinical symptoms are high fever with severe cough, and the child is less than 3 years old, the system includes a test kit for simultaneously detecting respiratory syncytial virus, adenovirus, Streptococcus pneumoniae, and metapneumovirus; When the child's clinical symptom is wheezing and the child is not less than 5 years old, the system includes a test kit for simultaneously detecting Mycoplasma pneumoniae, adenovirus, and Moraxella catarrhalis; When the child's clinical symptom is wheezing and the child is less than 5 years old, the system includes a test kit for simultaneously detecting respiratory syncytial virus, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae.

2. The use according to claim 1, characterized in that The children's respiratory pathogen detection kit is a fluorescent quantitative PCR detection kit.

3. The use according to claim 1, characterized in that The primers and probes for each pathogen in the detection system are as follows:

4. A kit for use according to any one of claims 1 to 3, characterized in that: The kit is used when the clinical symptom of the child is simple persistent high fever and the age is not less than 2 years old. The kit is a kit for detecting influenza A virus, influenza B virus, new coronavirus, and boca virus at the same time. The kit includes the following primers and probes: ， When the clinical symptom of the child is simple persistent high fever and the child is less than 2 years old, the system is a kit for simultaneously detecting influenza A virus, influenza B virus, new coronavirus, and enterovirus, and the kit includes the following primers and probes:

5. A kit for use according to any one of claims 1 to 3, characterized in that: The kit is used when the clinical symptom of the child is simple severe cough and the age is not less than 5 years old. The system is a kit for simultaneously detecting Mycoplasma pneumoniae, pertussis, metapneumovirus, and Moraxella catarrhalis; the kit includes the following primers and probes: ， When the child's clinical symptom is simple severe cough and the child is less than 5 years old, the system is a kit for simultaneously detecting rhinovirus, respiratory syncytial virus, adenovirus, and Mycoplasma pneumoniae; the kit includes the following primers and probes:

6. A kit for use according to any one of claims 1 to 3, characterized in that: The kit is used when the clinical symptoms of children are high fever with severe cough, and the age is not less than 3 years old. The kit is a kit for simultaneously detecting Mycoplasma pneumoniae, Streptococcus pneumoniae, parainfluenza virus, and Haemophilus influenzae. The kit includes the following primers and probes: ， When the clinical symptoms of children are high fever with severe cough, and the age is less than 3 years old, the system is a kit for simultaneously detecting respiratory syncytial virus, adenovirus, Streptococcus pneumoniae, and metapneumovirus, and the kit includes the following primers and probes:

7. A kit for use according to any one of claims 1 to 3, characterized in that: The kit is used when the clinical symptom of the child is wheezing and the age is not less than 5 years old. The kit is a kit for simultaneously detecting Mycoplasma pneumoniae, adenovirus, and Moraxella catarrhalis. The kit includes the following primers and probes: The kit is used when the clinical symptom of the child is wheezing and the child is less than 5 years old. The kit is a kit for simultaneously detecting respiratory syncytial virus, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae. The kit includes the following primers and probes:

Images